XRCC2, XRCC3 and RAD51 are the main molecules of the homologous recombinant repair (HRR) pathway related to thyroid cancer. Polymorphisms in these genes have been reported frequently in literature and are known to show diverse patterns in different populations. The present study was designed to screen these genes in thyroid cancer patients and controls at the DNA, mRNA and protein levels. A total of 456 pathologically confirmed thyroid cancer patients and 400 healthy controls were recruited. ARMS-PCR was used for genetic analysis followed by sequencing. In this study, various reported polymorphisms were analysed in HRR pathway genes at the germline level in thyroid cancer patients. Significant association of these SNPs were observed with age of diagnosis, gender, smoking, staging, histological subtype and treatment strategies of cancer. Significant association of these functional, promoter based and non-coding polymorphisms in HRR pathway genes and associations of these SNPs with important risk factors highlights their possible role in thyroid carcinogenesis. Further haplotype analysis revealed that most of the haplotypes in XRCC2, XRCC3 and RAD51 are linked with a significant increase in thyroid cancer risk. While some of the haplotypes were associated with a significant reduced thyroid cancer risk. RAD51 SNP, rs1801321 were observed consistent in reduced risk of thyroid cancer in association with risk factors analysed in this study. HRR pathway genes were further investigated at mRNA and protein expression levels. Thyroid cancer samples (n=102) along with equal numbers of un-involved tissues as controls were used for expression analysis. Quantitative real time PCR was used for determination of mRNA expression levels and immunohistochemistry was performed to analyse the protein expression of these genes. Expression analysis of XRCC2, XRCC3, RAD51 and proliferation marker Ki67 at mRNA level revealed significant deregulations. Significant downregulation (p<0.01) of XRCC2 and up-regulations of XRCC3 (p<0.01), RAD51 (p<0.001) and proliferation marker Ki67 (p<0.001) were observed in the expression profile of HRR molecules. These were significantly correlated (negatively and positively) with an up-regulated expression profile of the tumor proliferation marker, Ki67. XRCC3 and RAD51 expression was up-regulated in higher stages and aggressive tumor stages. Immunohistochemical analysis of HRR molecules revealed that among 102 tumor samples, 87% samples showed down- regulation of XRCC2 (p<0.0001), 75% samples showed up-regulation of XRCC3 (p<0.001), 76% samples showed up-regulation of RAD51 (p<0.0001) and 82% samples showed up-regulation (p<0.0001) of proliferation marker, Ki67. These IHC analysis results support our qPCR findings. Polymorphisms in HRR genes and abnormal expression at transcription and translation levels besides defective DNA damage may suggest that HRR pathway genes are correlated with thyroid tumorigenesis and aggressive proliferative behaviour of thyroid cancer in the Pakistani population. Furthermore, the role of XRCC2 and XRCC3 genes were explored in a thyroid cancer cell line (8505C) cells using in-vitro experiments. Effects of genetic modifications were observed for XRCC2 and XRCC3 in thyroid cancer cells by CRISPR-Cas9. These two genes were expressed using lentivirus having GFP, Cas9 and guide RNA. Gene knockouts for these genes were observed after single cell isolation. A gene cleavage assay showed successful genetic modifications and altered behaviour of these genes in thyroid cancer cells. Therefore, potential knockout of these genes gave an insight into molecular mechanism of thyroid cancer. XRCC3 gene function was further explored in the thyroid cancer cell line. Gene knockdown was produced by siRNA technology and successful knockdowns were confirmed using western blots. XRCC3 knockdown cells of anaplastic thyroid cancer (8505C) cell line showed decreased cell growth (p<0.01) as well as decreased cell proliferation after cell colony forming assay (p<0.001) when compared to controls. Decreased invasion (p<0.001), increased adhesion ability (p<0.001) and decreased migration abilities (i.e. wound closure for XRCC3 with siRNA knockdown was 86%) of the cells migrated for wound closure after 24 hours compared to ~98% cell migration of control cells. XRCC3 siRNA knockdown showed significant genetic alterations after treatment with topoisomerase inhibitor drugs (i.e. camptothecin, phleomycin and etoposide). Effect of these drugs on XRCC3 knockdowns were observed by colony PCR and metaphase analysis. Therefore overexpression of XRCC3 caused significant increase in cancerous characteristics of thyroid cancer which is in accordance with observed results at the mRNA and protein levels of expression. For a better insight into these genes in thyroid carcinogenesis, further work is needed to explore the interesting domains playing important role in carcinogenesis. Study of different factors affecting the thyroid cancer will not only assist in understanding thyroid cancer disease progression but will also help to establish future personalized cancer risk prediction.
Dr. Shari'ati is a revolutionary intellectual personality of this century. He regarded Islamic values as the guarantee of salvation and success for humanity. He sought to mobilize and revive frozen Islamic ideas. That is, tried to bring the Islamic concept out of the boundaries of formal and congested boundaries into common and general thoughts. Dr. Shari'ati also presented a unique view that divine Imam transcends than worldly governments and this divine leadership cannot be determined by (Shuraiet). Rather, it can be diagnosed by an obvious reason (Nass). This doctrine of Dr. Shari'ati is contrary to the ideology of the Sunni and the Shi'ite’s concept of Imamat and Khilafat because the Sunni sect believes that Khilafat Or Imamt should be determine by the Shurait (Council) and Shiites by the will(Nass). According to Dr. Shairathi, Imamat cannot be determining through Shourait or Nass but it can be identified by the superior attributes of the Imam. He believes that Imamat is not an external factor which can gain by attainment or by choice; rather, it is an Inherit object. In accepting this doctrine of Dr. Shari'ati, than the Imamat becomes a part of the system of naturalism (Takveeni). That makes the Imamat not a model process for humanity. So it would be a complicated issue to discuss and discover either the theory of Dr. Shari'ati is a applicable idea of Imamat or it is a just onlyu idialogy which cannot be practiced. The dissertation has been written to examine the reality of these two cases either Imamat is inherit case or it can be attainment case through Shouriat or Nass.
The present study highlights the possibility of using wastewater for forest irrigation. This study was conducted in 2 phases, first phase was the seed germination study of the five selected species and the second phase was of growth of plants through pot experiment. Five tree species selected for the study were, Dalbergia sissoo Roxb., Albizia lebbeck (L.) Benth, Pongamia pinnata (L.) Pierre, Bauhinia purpurea L. and Millettia peguensis Ali. Three types of wastewater were collected, first from industrial wastewater (IWW), second from hospital wastewater (HWW) and third from domestic wastewater (DWW). Germination experiment was set up in Petri dishes and seeds were irrigated with different concentrations of WWs. In pot experiment the five tree species were planted in pots and were irrigated with 5 different concentrations (0%, 25%, 50%, 75% and 100%) of WWs. Germination study results revealed that the five plant species survived in the WW irrigation and showed sufficient tolerance. The maximum germination was of Dalbergia sissoo i.e. 74%. The toxicity of different WWs is as follows: IWW > HWW > DWW The mean time to germination of Millettia peguensis was 95 hrs and of Albizia lebbeck was 80 hrs. Similarly, all the five species showed positive increase in seedling fresh weight, dry weight and seedling length. Maximum fresh weight was observed in Dalbergia sissoo (0.64 g) and the lowest fresh weight was reported in Millettia peguensis (0.35). The order of tolerance index and vigor index among the five species is as follows; Dalbergia sissoo > Albizia lebbeck > Bauhinia purpurea > Pongamia pinnata > Millettia peguensis In pot experiment, the DWW showed positive impact on height, fresh and dry weight, where as, the HWW showed positive impact on height upto 50% concentration and the further increase in concentration decreased the height. The maximum negative impact was observed with IWW irrigation. Pongamia pinnata showed 90% increase xiii in height in DWW compared to control set up. Albizia lebbeck showed 35% increase in fresh weight, 45% increase in fresh weight by Dalbergia sissoo. The photosynthetic rate, transpiration rate and stomatal conductance of all the five species decreased significantly. In HWW the decrease in photosynthetic rate was as follows; Pongamia pinnata (-80%), Albizia lebbeck (-60%), Dalbergia sissoo (-45%), Millettia peguensis (-45%) and Bauhinia purpurea (-58%). The proline content in all treatments was measured as a sign of oxidative stress. Maximum proline was observed in Bauhinia purpurea (6.33) in IWW, where as the least quantity of proline was observed in Pongamia pinnata (3.89). The metal uptake and translocation results are also very promising. Maximum uptake was observed for Pb in IWW by Dalbergia sissoo (107.06 mg/day). Uptake of Cr and Cu uptake was slow in all species. Translocation factor of Albizia lebbeck was maximum i.e. 3.03 in HWW. Untreated IWW seems to create number of problems in ecosystem by disturbing both biotic and abiotic (soil properties, soil osmotic potential) components. This study seems to be successful in combating wastewater problem. This study indicates that, Dalbergia sissoo, Albizia lebbeck, Bauhinia purpurea, Pongamia pinnata and Millettia peguensis are much tolerant in IWW and can be successfully used for phytoextraction processes. The tolerance index is as follows: Dalbergia sissoo > Albizia lebbeck > Bauhinia purpurea > Pongamia pinnata > Millettia peguensis The idea is to utilize WW to generate urban forests with the said five species. This idea can reduce multiple and serious problems like, IWW toxicity, WW treatment, and air pollution through urban forestry. The most prominent benefit is that this urban forest is eco-friendly and sustainable solution for multiple problems.